The Handel Lab

Research Areas

Experimental Methods

Chemokines and their Interactions

Chemokines are critical mediators of leukocyte cell migration during routine immune surveillance, lymphocyte development and homing, and inflammation [1, 2] . They function by binding to seven transmembrane G-protein coupled receptors (GPCRs) on target cells, causing conformational changes that trigger a cascade of intracellular signaling pathways involved in cell movement. Although chemokines were designed to carry out developmental and protective roles, they also have a dark side; specific chemokine receptors provide the portals for HIV to get into cells, while others contribute to inflammatory disease (e.g. multiple sclerosis, asthma, atherosclerosis, inflammatory bowel disease, graft rejection, and Alzheimer's disease) [3-5] . With respect to pathology, however, one of the most exciting findings from the last four years is that specific chemokines and receptors contribute to metastasis in many types of cancer [6-8] . Thus there is significant interest in developing strategies to antagonize their function, and in the case of cancer, a genuine opportunity to interfere with metastasis, the key process that causes death in most patients.

We have been using structural, biochemical, and more recently in vivo methods to understand the molecular details of chemokine-receptor interactions and function, and to identify ways to antagonize their function. In past and ongoing work, we focused on CCR2 and its MCP ligands, particularly MCP-1/CCL2, because of their reported roles in several inflammatory diseases including atherosclerosis [9, 10] and cancer [11, 12]. By carrying out detailed structural and mutagenic studies, we made several key discoveries: in short, that (i) MCP-1 binds its receptor as a monomer [13] ; (ii) we identified receptor binding, signaling and glycosaminoglycan (GAG) binding hotspots [14-16] ; (iii) we showed how receptor binding and signaling could be decoupled, thereby creating antagonists [15] ; (iv) using engineered mutants in experiments in vivo, we demonstrated that GAG-binding was critical for the function of MCP-1 and other chemokines [17] ; (v) we also showed with engineered mutants and in vivo experiments that chemokine oligomerization was critical for function [17] ; and (vi) we demonstrated that GAG-binding and oligomerization are functionally coupled, and proposed the concept that different oligomerization states may add to the specificity of GAG-binding, with important biological consequences like cellular localization [16-18] . From these types of detailed studies, several strategies to antagonize chemokine function have become apparent: (i) chemokine mutants that bind but do not activate their receptor; (ii and iii) GAG-binding deficient mutants and oligomerization deficient mutants; and (iv) GAGs themselves [19] .

After more than a decade of (ongoing) studies of the MCP-1/CCR2 system, we have begun to investigate other chemoattractant receptor systems. Within the chemokine family, there are ~ 45 human ligands and 18 receptors, and although many structure and mutagenic studies have been carried out, there are very few comprehensively studied systems, and most have not been studied at all. We have now expanded our research to other chemokine systems, particularly those involved in cancer metastasis [6-8] . The receptors are upregulated in many cancer cells, and their ligands are expressed in tissues that are the first metastatic destinations of these cells.

Our fundamental goals are to determine (1) the structure and biophysical properties of several of these proteins, and more recently, their membrane receptors; (2) how they bind their receptors and promote receptor-signaling, and what determines specificity in the interactions; (3) if and how they bind glycosaminoglycans (GAGs), and the potential role of oligomerization in these interactions; and (4) the sequences of high affinity GAG-binders, the extent of specificity in these chemoattractant:GAG interactions, and ultimately structural studies of chemoattractant:GAG complexes. (5) As strong proponents of protein and GAG-based therapeutics, in addition to generating fundamental information, we attempt to identify or design molecules that may be therapeutically useful, and in our health sciences environment at UCSD, push them into translational research studies. (6) Finally, we are characterizing the signaling properties of chemokines and receptors on cancer and other cells by genome and proteome expression profiling to add a true functional dimension to our work.

In addition to chemokines, we are also working on other non-chemokine chemoattractants, other cytokines, and viral "immunomodulatory" proteins that bind chemokines or other cytokines and block their action [20-21].

Relevant Papers

  1. Baggiolini, M, B Dewald, B Moser. "Human chemokines: an update." Annu Rev Immunol. 1997 15:675-705.
  2. Baggiolini, M. "Chemokines and leukocyte traffic." Nature. 1998 392:565-8.
  3. Baggiolini, M. "Chemokines in pathology and medicine." J Intern Med. 2001 250:91-104.
  4. Proudfoot, AE. "Chemokine receptors: multifaceted therapeutic targets." Nat Rev Immunol. 2002 2:106-15.
  5. Gerard, C, BJ Rollins. "Chemokines and disease." Nat Immunol. 2001 2:108-15.
  6. Muller, A, B Homey, H Soto, N Ge, D Catron, ME Buchanan, T McClanahan, E Murphy, W Yuan, SN Wagner, JL Barrera, A Mohar, E Verastegui, A Zlotnik. "Involvement of chemokine receptors in breast cancer metastasis." Nature. 2001 410:50-6.
  7. Zlotnik, A. "Chemokines and cancer." Ernst Schering Res Found Workshop. 2004 53-8.
  8. Murakami, T, AR Cardones, ST Hwang. "Chemokine receptors and melanoma metastasis." J Dermatol Sci. 2004 36:71-8.
  9. Boring, L, J Gosling, M Cleary, IF Charo. "Decreased lesion formation in CCR2-/- mice reveals a role for chemokines in the initiation of atherosclerosis. " Nature. 1998 394:894-7.
  10. Gosling, J, S Slaymaker, L Gu, S Tseng, CH Zlot, SG Young, BJ Rollins, IF Charo. "MCP-1 deficiency reduces susceptibility to atherosclerosis in mice that overexpress human apolipoprotein B." J Clin Invest. 1999 103:773-8.
  11. Ueno, T, M Toi, H Saji, M Muta, H Bando, K Kuroi, M Koike, H Inadera, K Matsushima. "Significance of macrophage chemoattractant protein-1 in macrophage recruitment, angiogenesis, and survival in human breast cancer" Clin Cancer Res. 2000 6:3282-9.
  12. Saji, H, M Koike, T Yamori, S Saji, M Seiki, K Matsushima, M Toi. "Significant correlation of monocyte chemoattractant protein-1 expression with neovascularization and progression of breast carcinoma." Cancer. 2001 92:1085-91.
  13. Paavola, CD, S Hemmerich, D Grunberger, I Polsky, A Bloom, R Freedman, M Mulkins, S Bhakta, D McCarley, L Wiesent, B Wong, K Jarnagin, TM Handel. "Monomeric monocyte chemoattractant protein-1 (MCP-1) binds and activates the MCP-1 receptor CCR2B." J Biol Chem. 1998 273:33157-65.
  14. Jarnagin, K, D Grunberger, M Mulkins, B Wong, S Hemmerich, C Paavola, A Bloom, S Bhakta, F Diehl, R Freedman, D McCarley, I Polsky, A Ping-Tsou, A Kosaka, TM Handel. "Identification of surface residues of the monocyte chemotactic protein 1 that affect signaling through the receptor CCR2." Biochemistry. 1999 38:16167-77.
  15. Hemmerich, S, C Paavola, A Bloom, S Bhakta, R Freedman, D Grunberger, J Krstenansky, S Lee, D McCarley, M Mulkins, B Wong, J Pease, L Mizoue, T Mirzadegan, I Polsky, K Thompson, TM Handel, K Jarnagin. "Identification of residues in the monocyte chemotactic protein-1 that contact the MCP-1 receptor, CCR2." Biochemistry. 1999 38:13013-25.
  16. Lau, EK, CD Paavola, Z Johnson, JP Gaudry, E Geretti, F Borlat, AJ Kungl, AE Proudfoot, TM Handel. "Identification of the glycosaminoglycan binding site of the CC chemokine, MCP-1: implications for structure and function in vivo." J Biol Chem. 2004 279:22294-305.
  17. Proudfoot, AEI, TM Handel, Z Johnson, EK Lau, P LiWang, I Clark-Lewis, F Borlat, TNC Wells, MH Kosco-Vilbois. "Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines." PNAS. 2003 100:1885-90.
  18. Lau, EK, S Allen, AR Hsu, TM Handel. "Chemokine-receptor interactions: GPCRs, glycosaminoglycans and viral chemokine binding proteins." Adv Protein Chem. 2004 68:351-91.
  19. Johnson, Z, AEI Proudfoot, TM Handel. "Interaction of chemokines and glycosaminoglycans: A new twist in the regulation of chemokine function with opportunities for therapeutic intervention" Cytokine and Growth Factor Reviews. 2005
  20. Alexander, J.M. C.A. Nelson, V. van Berkel Fezzie, E.K. Lau, J.M. Studts, T.J. Brett, S.H. Speck, T.M. Handel, H.W. Virgin and D.H. Fremont "Structural basis of chemokine sequestration by a herpesvirus decoy receptor." Cell. 2002 111:343-56.
  21. Seet, B.T., C.A. McCaughan, T.M. Handel, G. McFadden and S. B. Fleming. "Identification and analysis of a chemokine binding protein from the orf poxvirus." PNAS. 2003 100:15137-42.

Immunomodulatory Proteins Involved in Apoptosis

Under construction.

Protein Design

Alongside our more traditional structural and biochemical methods, we also take an engineering approach. Over the last decade, techniques have evolved to either improve current or produce novel functions in proteins. Directed evolution and catalytic antibodies are the first examples that come to mind. While both of these techniques harness the power of evolution, they lack physical insight into the reasons behind the mutations that occur.

"Rational" design is distinct from these techniques in that it attempts to choose mutations based on a physical understanding of the system. Such design is useful because success or failure is an indicator of the accuracy of our knowledge of the system. This is especially important for proteins, where the basic physical principles that govern their structure are still under debate.

Computational protein design takes rational design a step further, attempting to automate the process. By developing an accurate energy function that is able to quickly approximate the relevant physics of a protein system, we can highlight mutations that may have the desired effect. With search algorithms such as Monte Carlo and Dead-End-Elimination, we can find reasonable minima in the design space.

The field of protein design may not be young, but its potential is still mostly untapped. Computational design allows us to expand the applications of protein design to systems that were previously intractable by directed evolution or catalytic antibodies. While it may not be as effective as simple evolution in all cases, it is potentially more widely applicable and teaches us more about the true, underlying physics.

Relevant Papers